This project deploys a range of structural techniques to examine normal synaptic structure. These approaches have in common their dependence on rapid freezing and direct visualization of living brain by light microscopic techniques. Up until now this project has been engaged in explorations of various live brain preparations suitable for these purposes. Recently, an isolated whole brain preparation maintained in vitro by vascular perfusion as well superfusion with artificial cerebrospinal fluid (CSF) as been evaluated. The ultrastructure of surface samples of isolated brains rapidly excised, quick frozen and freeze-substituted served as a benchmark to choose a perfusion fixative yielding realistic images of synaptic structures. It could now be determined to what extent deeper cortical regions of perfused brains remained structurally intact. Throughout 2 hours of perfusion, the morphology of synaptic structures in the isolated brain remained equivalent to the normal brain perfused-fixed in situ. These results provide an excellent method for structural work on the isolated brain, and show that the isolated brain can be used for studies of synaptic structure depending on rapid freeze fixation, and are in agreement with the reported persistence of electrophysiological functions in this preparation. (A comprehensive effort to develop methods for making and maintaining organotypic brain cultures continues and now appears to be successful - Project Z01 NS 02610-11 LN). Thus, these results will be reported but the main future emphasis will be on organotypic cultures.